ORIGINAL RESEARCH published: 28 January 2016 doi: 10.3389/fmicb.2016.00028 Metagenomic Analysis Indicates Epsilonproteobacteria as a Potential Cause of Microbial Corrosion in Pipelines Injected with Bisulfite Dongshan An 1, Xiaoli Dong 2, Annie An 1, Hyung S. Park 1, Marc Strous 2 and Gerrit Voordouw 1* 1 Petroleum Microbiology Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB, Canada, 2 Department of Geosciences, University of Calgary, Calgary, AB, Canada Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in Edited by: pipelines transporting brackish subsurface water used in the production of bitumen Wael Ahmed Ismail, Arabian Gulf University, Bahrain by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has Reviewed by: indicated that SBS addition increased the fraction of the sulfate-reducing bacteria Christopher L. Hemme, (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by University of Rhode Island, USA Jan Kuever, disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed Bremen Institute for Materials Testing, by reduction of aqueous protons at the iron surface, or use low potential electrons Germany from iron and aqueous protons directly for sulfate reduction. In order to reveal the Huda Mahmoud Mahmoud, Kuwait University, Kuwait effects of SBS treatment in more detail, metagenomic analysis was performed with *Correspondence: pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and Gerrit Voordouw downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in [email protected] microbial community composition and in affiliated hynL genes for the large subunit of Specialty section: [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of This article was submitted to the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize Microbiotechnology, Ecotoxicology and Bioremediation, sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely 0 a section of the journal catalyzed reduction of sulfur (S ) originating from the metabolism of bisulfite with cathodic Frontiers in Microbiology H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel Received: 17 November 2015 (Fe0). Overall this accelerates reaction of of S0 and Fe0 to form FeS, making this class Accepted: 11 January 2016 Published: 28 January 2016 a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome Citation: also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium An D, Dong X, An A, Park HS, and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Strous M and Voordouw G (2016) Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating Metagenomic Analysis Indicates Epsilonproteobacteria as a Potential anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger Cause of Microbial Corrosion in function. Pipelines Injected with Bisulfite. Front. Microbiol. 7:28. Keywords: corrosion, pipeline, microbiologically influenced corrosion (MIC), microbial community analysis, doi: 10.3389/fmicb.2016.00028 metagenomics, sulfate reducing bacteria (SRB), Epsilonproteobacteria, hydrogenase Frontiers in Microbiology | www.frontiersin.org 1 January 2016 | Volume 7 | Article 28 An et al. Metagenomics of Internal Pipeline Corrosions INTRODUCTION in EMIC involves formation of H2 both types of enzymes may contribute. In addition to SRB, hydrogenotrophic methanogens Pipeline failure caused by corrosion can have serious and acetogens have been found to contribute to MIC by using consequences for the oil and gas industry (Ossai et al., 2015) and cathodic H2 or anodic electrons for reduction of CO2 to methane understanding the causes of corrosion is therefore important. and acetate, respectively (Dinh et al., 2004; Mand et al., 2014). While chemical reaction between oxygen and iron is the main As indicated previously (Park et al., 2011), the presence of cause of external pipeline corrosion, microbiologically influenced MIC-causing SRB can be promoted by injection of sodium corrosion (MIC) under mostly anoxic conditions can account bisulfite (SBS), which is used as an oxygen scavenger to for up to 40% of internal pipeline corrosion in the oil and gas decrease oxygen-mediated corrosion in pipelines and other industry (Zhu et al., 2003). The application of high-throughput steel infrastructure. Injection of SBS into pipelines transporting sequencing technologies has indicated that diverse microbes are brackish subsurface water to a plant generating steam for involved in internal pipeline corrosion, such as sulfate reducing production of bitumen by steam-assisted gravity drainage caused bacteria (SRB), acid-producing fermentative bacteria, including a drastic change in microbial community composition of acetogens, as well as methanogens (Dinh et al., 2004; Park et al., pipe-associated solids (PAS). Relative to solids from a pipe 2011; Mand et al., 2014; Okoro et al., 2014; Yang et al., 2014). section upstream of the SBS injection point (PAS-616P), solids Among these SRB are often considered to be the major MIC from a downstream pipe section (PAS-821TP) had a smaller causative agents. fraction of methanogens of the family Methanobacteriaceae and Fundamentally all SRB can corrode iron indirectly by larger fractions of SRB of the genera Desulfomicrobium and producing the corrosive chemical agent hydrogen sulfide (H2S). Desulfocapsa (Park et al., 2011). Desulfocapsa can also grow This has been referred to as “chemical microbially-influenced by disproportionating bisulfite into sulfide and sulfate (Finster, corrosion” (CMIC) by Enning and Garrelfs (2014). H2S is 2008). Here we evaluate the genetic potential of the microbial produced during sulfate reduction by SRB with electrons usually communities in these two PAS samples in more detail by an in derived from organic acids, alcohols, or hydrogen (H2), which depth metagenomic analysis with a focus on hydrogenase genes. is formed by fermentation of organic compounds in anoxic settings (Muyzer and Stams, 2008). In the absence of organic MATERIALS AND METHODS electron donors and in the presence of metallic iron, SRB may obtain energy from oxidation of cathodic H2 formed by chemical Sample Collection reaction between protons from water and electrons from anodic Two cutouts from a brackish water-transporting pipeline system dissolution of iron, accelerating corrosion (Mand et al., 2014). were collected upstream (616P) and downstream (821TP) from Whether SRB are capable of accelerating corrosion by scavenging the SBS injection point. These were the same as described cathodic H2, which was proposed long ago (Von Wolzogen Kühr elsewhere (Park et al., 2011). The pipeline cutouts were immersed and Van der Vlugt, 1934), is still controversial (Enning and in pipe-associated water (PAW) from the site, were shipped Garrelfs, 2014). Instead, some SRB are thought to corrode iron in sealed, airtight buckets and received in the lab within 24 h. through direct uptake of the anodic electrons with protons from The cutouts and the associated waters were then immediately water for sulfate reduction (“electrical microbially-influenced transferred to a Coy anaerobic hood with an atmosphere of 90% corrosion”; EMIC; Enning and Garrelfs, 2014). (v/v) N2 and 10% CO2. PAS-616P and PAS-821TP were obtained The microbial consumption or production of H2 is by scraping the drained surface of the cutouts with a sterile catalyzed by hydrogenases that can be divided into two spatula. These were then re-suspended in 260 mL of PAW-616P main phylogenetically unrelated groups, the [NiFe]- and [FeFe]- and PAW-821TP, respectively, filtered using an 0.2 µm Millipore hydrogenases (Vignais and Billoud, 2007). [NiFe]-hydrogenases filter (Nylon membrane, USA) prior to use. are common in Archaea and Bacteria. Many are external to the Chemical analyses conducted on the samples included the cytoplasm and are primarily associated with H2 oxidation in measurement of pH, sulfide (Trüper and Schlegel, 1964), oxic and anoxic metabolism. [FeFe]-hydrogenases are found in sulfate (ion chromatography with conductivity detector/anion Bacteria and Eukarya. These enzymes are especially prevalent column), ammonium, nitrite (ion chromatography with UV in the cytoplasm of anaerobic fermentative organisms (e.g., the detector/anion column), and organic acids (ion chromatography Firmicutes), where they form hydrogen in metabolic reactions with UV detector/organic acids column), as detailed else where coupling the oxidation of reduced electron carriers (NADH, (Park et al., 2011). NADPH or reduced ferredoxin, Fdred) to the reduction of protons. SRB of the genus Desulfovibrio are exceptional in DNA Isolation having a periplasmic [FeFe]-hydrogenase, which functions in DNA was extracted from the PAS samples using a bead-beating hydrogen oxidation at high hydrogen concentration (Caffrey procedure outlined by the manufacturer of the FastDNAR Spin et al., 2007). Hence, assuming that consumption of cathodic Kit for Soil (MP Biomedicals). The extracted DNA was further hydrogen is important in MIC, both types of hydrogenases purified by cesium chloride density gradient centrifugation. can contribute. However, [NiFe]-hydrogenases may be more The concentration of DNA was quantified using the Qubit important than